Polyketide synthases (PKSs) are a family of multi-enzyme assemblies that catalyze the biosynthesis of numerous structurally complex and biologically important natural products. Bacterial aromatic PKSs are one subclass of PKSs responsible for the biosynthesis of natural products such as doxorubicin and tetracycline. They are composed of 3-10 distinct subunits, which together synthesize a polyfunctional aromatic product. The modularity of these multi-enzyme systems has been exploited via genetic engineering for the biosynthesis of numerous "unnatural" natural products. However, our understanding of the structural and mechanistic principles by which these remarkable enzymes assemble and catalyze multi-step transformations involving highly reactive intermediates is rudimentary. During the past proposal period we have expressed, purified, and reconstituted the actinorhodin (act), tetracenomycin (tcm), and parts of the R1128 PKSs. This has allowed us to probe the properties of selected aromatic PKS components using a combination of mutagenesis, protein chemical, structural (NMR and X-ray crystallography), and biosynthetic engineering approaches. The specific goals for the next proposal period are: 1) Development of improved expression systems, purification procedures, and assay systems for aromatic PKSs and their components, 2) Further structural and mechanistic analysis, and biosynthetic exploitation of the unusual primer unit tolerance of the R1128 PKS, 3) Further structural and mechanistic analysis, and engineering of the chain length specificity of the act, tcm, and possibly other minimal PKSs, 4) Dissecting and engineering the malonyl-CoA selectivity of the malonyl-CoA:acyl carrier protein acyltransferase, 5) Analyzing the kinetic consequences of protein-protein interactions between the ketosynthase, acyl carrier protein, and acyltransferase, and 6) Engineering a hybrid PKS that contains components of an aromatic and a modular PKS. These studies, which follow logically from our results thus far, are expected to provide interesting and important insights into structure-function relationships within this remarkable family of multi-enzyme assemblies. Furthermore, knowledge acquired in the process could expand the utility of bacterial aromatic PKSs (and possibly other PKSs as well) for the engineered biosynthesis of novel natural products.